Our long-term goal is the discovery of methods for achieving islet transplantation tolerance for the treatment of diabetes. During the previous funding period, we developed a tolerance induction protocol consisting of a single donor specific transfusion (DST) and a brief course of anti-CD154 mAb and with it achieved permanent islet allograft survival in diabetic mice. But as we contemplate translation of this or a comparable protocol to the clinic, our knowledge base in two key areas remains limited. First, mechanisms underlying the induction of tolerance are only partially understood. Second, little is known about mechanisms underlying the maintenance of tolerance to successful grafts. To gain the needed knowledge, we have created three hematopoietic "synchimeras" that circulate trace levels of "marked" alloreactive transgenic T cells in syngeneic but genetically unaltered mice. These systems permit us to determine the fate of T cells with known alloreactive specificity in the presence of a normal immune system. The synchimera model systems will permit us to discover the cellular mechanisms underlying both the induction and maintenance of allograft tolerance--revealing the fate of existing alloreactive CD4 + and CD8 + T cells as they are tolerized and then the fate of newly developed alloreactive cells that emigrate from the thymus of hosts beating intact allografts. These tools will also allow us to investigate the role of both direct and indirect antigen presentation not only in allograft rejection but also in tolerance. Our synchimeric models will be used to address two Specific Aims. Specific Aim 1 is to identify mechanisms responsible for induction of transplantation tolerance in CD4+ and CD8 + alloreactive TCR transgenic synchimeric mice. In this aim we will test the hypothesis that tolerance induction requires deletion of the majority of CD4 + and CD8 + alloreactive T cells. Specific Aim 2 is to identify mechanisms responsible for maintenance of transplantation tolerance in CD4 + and CD8 + alloreactive TCR transgenic synchimeras. We will test the hypothesis that maintenance of allografi tolerance requires the presence of regulatory CD4 + T cells and the hypothesis that critical regulatory CD4 + T cell populations required for allograft maintenance are derived from alloreactive CD4 + T cell populations that escape deletion and become anergic. It is our belief that the information gained will 1) identify the cellular mechanism that controls the induction and maintenance of transplantation tolerance and 2) be applicable to the development of newer and better tolerance-based islet transplantation strategies to cure human diabetes.